Shear-stress-induced von Willebrand factor binding to platelets causes the activation of tyrosine kinase(s)

Pathological arterial blood flow generates fluid shear stresses that directly cause platelet aggregation. The mechanism of shear-induced platelet aggregation is incompletely understood, but involves von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib and GP IIb-IIIa, leading to the transmembrane influx of Ca²⁺ and the activation of protein kinase C. To investigate this further, shear-stress-induced protein tyrosine phosphorylation (PTP) of washed platelets was studied in a cone-plate viscometer. A time- and shear-stress-dependent tyrosine phosphorylation of substrates with approx. M(r) 29000-31000, 36000, 50000, 58000, 64000, 76000, 85000 and 105000 was observed. PTP in response to a threshold shear stress of 0.3 mN/ cm² (30 dyn/cm²) was enhanced in most cases by exogenous purified human vWF, and PTP in response to a pathological shear stress of 0.9 mN/cm² (90 dyn/cm²) was inhibited in some cases by inhibiting vWF binding to GP Ib or GP IIb-IIIa, or by inhibiting Ca²⁺ responses with extracellular EGTA. Shear-induced PTP of a substrate of M(r)≃31000 appeared to be independent of GP Ib, and PTP of a substrate(s) of M(r)≃29000 was shear-stress-dependent but independent of extracellular Ca²⁺. Cytochalasin D, which inhibits GP Ib-cytoskeleton interactions, inhibits the PTP of a substrate of M(r)≃76000. These results suggest that tyrosine phosphorylation may be involved in transmembrane signalling that mediates platelet adhesion and aggregation in response to pathological shear stresses generated at sites of arterial vaso-occlusion.